U.S. patent number 8,354,477 [Application Number 12/849,683] was granted by the patent office on 2013-01-15 for multi-armed, monofunctional, and hydrolytically stable derivatives of poly(ethylene glycol) and related polymers for modification of surfaces and molecules.
This patent grant is currently assigned to Nektar Therapeutics. The grantee listed for this patent is Paolo Caliceti, J. Milton Harris, Oddone Schiavon, Francesco Maria Veronese. Invention is credited to Paolo Caliceti, J. Milton Harris, Oddone Schiavon, Francesco Maria Veronese.
United States Patent |
8,354,477 |
Harris , et al. |
January 15, 2013 |
Multi-armed, monofunctional, and hydrolytically stable derivatives
of poly(ethylene glycol) and related polymers for modification of
surfaces and molecules
Abstract
Multi-armed, monofunctional, and hydrolytically stable polymers
are described having the structure ##STR00001## wherein Z is a
moiety that can be activated for attachment to biologically active
molecules such as proteins and wherein P and Q represent linkage
fragments that join polymer arms poly.sub.a and poly.sub.b,
respectively, to central carbon atom, C, by hydrolytically stable
linkages in the absence of aromatic rings in the linkage fragments.
R typically is hydrogen or methyl, but can be a linkage fragment
that includes another polymer arm. A specific example is an mPEG
disubstituted lysine having the structure ##STR00002## where
mPEG.sub.a and mPEG.sub.b have the structure
CH.sub.3O--(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2-- wherein n
may be the same or different for poly.sub.a- and poly.sub.b- and
can be from 1 to about 1,150 to provide molecular weights of from
about 100 to 100,000.
Inventors: |
Harris; J. Milton (Huntsville,
AL), Veronese; Francesco Maria (Padua, IT),
Caliceti; Paolo (Padua, IT), Schiavon; Oddone
(Padua, IT) |
Applicant: |
Name |
City |
State |
Country |
Type |
Harris; J. Milton
Veronese; Francesco Maria
Caliceti; Paolo
Schiavon; Oddone |
Huntsville
Padua
Padua
Padua |
AL
N/A
N/A
N/A |
US
IT
IT
IT |
|
|
Assignee: |
Nektar Therapeutics (San
Francisco, CA)
|
Family
ID: |
27005224 |
Appl.
No.: |
12/849,683 |
Filed: |
August 3, 2010 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20100298496 A1 |
Nov 25, 2010 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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12284357 |
Sep 18, 2008 |
7786221 |
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10119546 |
Apr 10, 2002 |
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09939867 |
Aug 27, 2001 |
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09140907 |
Aug 27, 1998 |
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08443383 |
May 17, 1995 |
5932462 |
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08371065 |
Jan 10, 1995 |
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Current U.S.
Class: |
525/419; 435/129;
514/424; 435/181; 435/177; 435/180; 435/188; 525/425 |
Current CPC
Class: |
C08G
65/48 (20130101); C08G 65/329 (20130101); A61K
47/60 (20170801); C12N 9/96 (20130101); Y10S
530/815 (20130101); Y10S 530/816 (20130101) |
Current International
Class: |
C08G
65/34 (20060101); C08F 283/00 (20060101) |
Field of
Search: |
;525/419,425 ;514/424
;435/188,94.3,177,180,181 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0400472 |
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Dec 1990 |
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EP |
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0400486 |
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Dec 1990 |
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EP |
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0473084 |
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Mar 1992 |
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EP |
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0632082 |
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Jan 1995 |
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EP |
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WO 94/26778 |
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Nov 1994 |
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WO |
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WO 95/11924 |
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May 1995 |
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WO |
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Other References
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applicant .
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Applications, edited by J. Milton Harris, Plenum Press, New York
(1992), Chapter 9, pp. 127-137 (1992). cited by applicant .
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Glycol) and Its Application in Surface Proteins Modification," XVII
Congresso Nazionale della Societa Chimica Italiana, Januachem 92,
Genova Oct. 25-30, 1992, pp. 300-301 (1992). cited by applicant
.
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Asparaginase," National Academy of Sciences, 613:95-108 (1990).
cited by applicant .
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Modification of Proteins," Agric. Biol. Chem., 52(8):2125-2127
(1998). cited by applicant .
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Methoxypolyethylene Glycol Derivative," Agric. Biol. Chem.,
54(10):2635-2640 (1990). cited by applicant .
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Attachment of Polyethylene Glycol to Proteins," Biotechnology and
Applied Biochemistry, 15:100-114 (1992). cited by applicant .
Zalipsky et al., "Succinimidyl Carbonates of Polyethylene Glycol:
Useful Reactive Polymers for Preparation of Protein Conjugates,"
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applicant .
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applicant.
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Primary Examiner: Harlan; Robert D.
Attorney, Agent or Firm: Evans; Susan T. Wilson; Mark A.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser.
No. 12/284,357, filed Sep. 18, 2008, now U.S. Pat. No. 7,786,221,
which is a continuation of U.S. patent application Ser. No.
10/119,546, filed Apr. 10, 2002, now abandoned, which is a
continuation of U.S. patent application Ser. No. 09/939,867, filed
Aug. 27, 2001, now abandoned, which is a continuation of U.S.
patent application Ser. No. 09/140,907, filed Aug. 27, 1998, now
abandoned, which is a continuation of U.S. patent application Ser.
No. 08/443,383, filed May 17, 1995, now U.S. Pat. No. 5,932,462,
which is a continuation-in-part of U.S. patent application Ser. No.
08/371,065, filed Jan. 10, 1995, now abandoned, which are hereby
incorporated by reference herein in their entireties.
Claims
What is claimed is:
1. A method for preparing a conjugate of a purified branched
water-soluble polymer, comprising: a. providing an impure polymer
composition comprising (i) a branched water-soluble polymer having
the structure: ##STR00039## where Z is a moiety comprising a site
suitable for interacting with ion exchange chromatography media,
and mPEG.sub.a and mPEG.sub.b are each independently a methoxy
polyethylene glycol, and (ii) one or more polymeric impurities
selected from the group consisting of PEG diol, mPEG-OH, and
activated mPEG, b. purifying the impure polymer composition by ion
exchange chromatography under conditions effective to essentially
remove the polymeric impurities to thereby provide a purified
branched water-soluble polymer that is in essentially pure form, c.
optionally reacting the purified branched water-soluble polymer
with a reagent effective to transform Z to a Z-activated moiety
suitable for reaction with a nucleophilic group of a biologically
active molecule, and d. reacting the purified branched water
soluble polymer of step b. or step c. with one or more of the
nucleophilic groups of the biologically active molecule under
conditions effective to form a conjugate of the purified branched
water soluble polymer and the biologically active molecule.
2. The method of claim 1 wherein Z is carboxyl.
3. The method of claim 1, wherein said polymeric impurities further
comprise a mono-substituted mPEG intermediate.
4. The method of claim 3, wherein the mono-substituted mPEG
intermediate is mPEG-mono-substituted lysine.
5. The method of claim 1, wherein said purifying further comprises:
loading the impure polymer composition onto an ion exchange
chromatography medium to provide a loaded medium, washing the
polymeric impurities from said loaded medium using an aqueous
eluent under conditions effective to elute said impurities from
said medium, adjusting the conditions of the aqueous eluent to
effect elution of said branched water-soluble polymer from the
medium, eluting said branched water-soluble polymer from said
medium to provide an aqueous solution comprising the purified
branched water-soluble polymer in essentially pure form, and
recovering the purified branched water-soluble polymer from said
aqueous solution.
6. The method of claim 1, wherein said branched water soluble
polymer has a molecular weight ranging from about 10,000 daltons to
about 50,000 daltons.
7. The method of claim 6, wherein the branched water soluble
polymer has a molecular weight of about 40,000 daltons.
8. The method of claim 1, wherein mPEG.sub.a and mPEG.sub.b are the
same.
9. The method of claim 1 including step c., wherein the Z-activated
moiety is an active ester.
10. The method of claim 9, wherein the active ester is a
succinimidyl ester.
11. The method of claim 1, including step c., wherein the
Z-activated moiety is selected from the group consisting of
trifluoroethylsulfonate, isocyanate, isothiocyanate, active
carbonate, aldehyde, sulfone, vinyl sulfone, malemide,
iodoacetamide, and iminoester.
12. method of claim 11, wherein the Z-activated moiety is an active
carbonate selected from succinimidyl carbonate,
p-nitrophenylcarbonate, and trichlorophenylcarbonate.
13. The method of claim 1, wherein the biologically active molecule
is selected from the group consisting of a peptide, a protein, a
nucleotide, a polynucleotide, a lipid, and a small molecule
drug.
14. The method of claim 1, wherein the nucleophilic group on the
biologically active molecule is selected from amino, thiol, and
hydroxyl.
15. The method of claim 14, wherein the nucleophilic group on the
biologically active molecule is amino.
16. The method of claim 1, wherein the biologically active molecule
is an interferon.
17. The method of claim 16, wherein the interferon is an
interferon-.alpha..
18. The method of claim 17, wherein the nucleophilic group is an
amino group.
19. A method according to claim 1 including step c., wherein: (i)
the branched water soluble polymer in a.(i) is a mPEG-disubstituted
lysine branched polymer having the structure: ##STR00040## (ii) the
one or more polymeric impurities are selected from the group
consisting of PEG diol, mPEG-OH, activated mPEG, and
mPEG-mono-substituted lysine, (iii) the --COOH group of the
purified mPEG-disubstituted lysine is transformed to an activated
ester, and (iv). step d. comprises reacting the purified activated
ester of step c. with amino groups of an interferon molecule under
conditions effective to form an interferon mPEG-disubstituted
lysine conjugate.
Description
FIELD OF THE INVENTION
This invention relates to monofunctional derivatives of
poly(ethylene glycol) and related polymers and to methods for their
synthesis and activation for use in modifying the characteristics
of surfaces and molecules.
BACKGROUND OF THE INVENTION
Improved chemical and genetic methods have made many enzymes,
proteins and other peptides and polypeptides available for use as
drugs or biocatalysts having specific catalytic activity. However,
limitations exist to use of these compounds.
For example, enzymes that exhibit specific biocatalytic activity
sometimes are less useful than they otherwise might be because of
problems of low stability and solubility in organic solvents.
During in vivo use, many proteins are cleared from circulation too
rapidly. Some proteins have less water solubility than is optimal
for a therapeutic agent that circulates through the bloodstream.
Some proteins give rise to immunological problems when used as
therapeutic agents. Immunological problems have been reported
manufactured proteins even where the compound apparently has the
same basic structure as the homologous natural product. Numerous
impediments to the successful use of enzymes and proteins as drugs
and biocatalysts have been encountered.
One approach to the problems that have arisen in the use of
polypeptides as drugs or biocatalysts has been to link suitable
hydrophilic or amphiphilic polymer derivatives to the polypeptide
to create a polymer cloud surrounding the polypeptide. If the
polymer derivative is soluble and stable in organic solvents, then
enzyme conjugates with the polymer may acquire that solubility and
stability. Biocatalysis can be extended to organic media with
enzyme and polymer combinations that are soluble and stable in
organic solvents.
For in vivo use, the polymer cloud can help to protect the compound
from chemical attack, to limit adverse side effects of the compound
when injected into the body, and to increase the size of the
compound, potentially to render useful compounds that have some
medicinal benefit, but otherwise are not useful or are even harmful
to an organism. For example, the polymer cloud surrounding a
protein can reduce the rate of renal excretion and immunological
complications and can increase resistance of the protein to
proteolytic breakdown into simpler, inactive substances.
However, despite the benefits of modifying polypeptides with
polymer derivatives, additional problems have arisen. These
problems typically arise in the linkage of the polymer to the
polypeptide. The linkage may be difficult to form. Bifunctional or
multifunctional polymer derivatives tend to cross link proteins,
which can result in a loss of solubility in water, making a
polymer-modified protein unsuitable for circulating through the
blood stream of a living organism. Other polymer derivatives form
hydrolytically unstable linkages that are quickly destroyed on
injection into the blood stream. Some linking moieties are toxic.
Some linkages reduce the activity of the protein or enzyme, thereby
rendering the protein or enzyme less effective.
The structure of the protein or enzyme dictates the location of
reactive sites that form the loci for linkage with polymers.
Proteins are built of various sequences of alpha-amino acids, which
have the general structure
##STR00003## The alpha amino moiety (H.sub.2N--) of one amino acid
joins to the carboxyl moiety (--COOH) of an adjacent amino acid to
form amide linkages, which can be represented as
##STR00004## where n can be hundreds or thousands. The terminal
amino acid of a protein molecule contains a free alpha amino moiety
that is reactive and to which a polymer can be attached. The
fragment represented by R can contain reactive sites for protein
biological activity and for attachment of polymer.
For example, in lysine, which is an amino acid forming part of the
backbone of most proteins, a reactive amino (--NH.sub.2) moiety is
present in the epsilon position as well as in the alpha position.
The epsilon --NH.sub.2 is free for reaction under conditions of
basic pH. Much of the art has been directed to developing polymer
derivatives having active moieties for attachment to the epsilon
--NH.sub.2 moiety of the lysine fraction of a protein. These
polymer derivatives all have in common that the lysine amino acid
fraction of the protein typically is modified by polymer
attachment, which can be a drawback where lysine is important to
protein activity.
Poly(ethylene glycol), which is commonly referred to simply as
"PEG," has been the nonpeptidic polymer most used so far for
attachment to proteins. The PEG molecule typically is linear and
can be represented structurally as
HO--(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2--OH or, more simply,
as HO-PEG-OH. As shown, the PEG molecule is difunctional, and is
sometimes referred to as "PEG diol." The terminal portions of the
PEG molecule are relatively nonreactive hydroxyl moieties, --OH,
that can be activated, or converted to functional moieties, for
attachment of the PEG to other compounds at reactive sites on the
compound.
For example, the terminal moieties of PEG diol have been
functionalized as active carbonate ester for selective reaction
with amino moieties by substitution of the relatively nonreactive
hydroxyl moieties, --OH, with succinimidyl active ester moieties
from N-hydroxy succinimide. The succinimidyl ester moiety can be
represented structurally as
##STR00005## Difunctional PEG, functionalized as the succinimidyl
carbonate, has a structure that can be represented as
##STR00006##
Difunctional succinimidyl carbonate PEG has been reacted with free
lysine monomer to make high molecular weight polymers. Free lysine
monomer, which is also known as alpha, epsilon diaminocaproic acid,
has a structure with reactive alpha and epsilon amino moieties that
can be represented as
##STR00007##
These high molecular weight polymers from difunctional PEG and free
lysine monomer have multiple, pendant reactive carboxyl groups
extending as branches from the polymer backbone that can be
represented structurally as
##STR00008##
The pendant carboxyl groups typically have been used to couple
nonprotein pharmaceutical agents to the polymer. Protein
pharmaceutical agents would tend to be cross linked by the
multifunctional polymer with loss of protein activity.
Multiarmed PEGS having a reactive terminal moiety on each branch
have been prepared by the polymerization of ethylene oxide onto
multiple hydroxyl groups of polyols including glycerol. Coupling of
this type of multi-functional, branched PEG to a protein normally
produces a cross-linked product with considerable loss of protein
activity.
It is desirable for many applications to cap the PEG molecule on
one end with an essentially nonreactive end moiety so that the PEG
molecule is monofunctional. Monofunctional PEGS are usually
preferred for protein modification to avoid cross linking and loss
of activity. One hydroxyl moiety on the terminus of the PEG diol
molecule typically is substituted with a nonreactive methyl end
moiety, CH.sub.3--. The opposite terminus typically is converted to
a reactive end moiety that can be activated for attachment at a
reactive site on a surface or a molecule such as a protein.
PEG molecules having a methyl end moiety are sometimes referred to
as monomethoxy-polyethylene glycol) and are sometimes referred to
simply as "mPEG." The mPEG polymer derivatives can be represented
structurally as
H.sub.3C--O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--Z where n
typically equals from about 45 to 115 and --Z is a functional
moiety that is active for selective attachment to a reactive site
on a molecule or surface or is a reactive moiety that can be
converted to a functional moiety.
Typically, mPEG polymers are linear polymers of molecular weight in
the range of from about 1,000 to 5,000. Higher molecular weights
have also been examined, up to a molecular weight of about 25,000,
but these mPEGs typically are not of high purity and have not
normally been useful in PEG and protein chemistry. In particular,
these high molecular weight mPEGs typically contain significant
percentages of PEG diol.
Proteins and other molecules typically have a limited number and
distinct type of reactive sites available for coupling, such as the
epsilon --NH.sub.2 moiety of the lysine fraction of a protein. Some
of these reactive sites may be responsible for a protein's
biological activity. A PEG derivative that attached to a sufficient
number of such sites to impart the desired characteristics can
adversely affect the activity of the protein, which offsets many of
the advantages otherwise to be gained.
Attempts have been made to increase the polymer cloud volume
surrounding a protein molecule without further deactivating the
protein. Some PEG derivatives have been developed that have a
single functional moiety located along the polymer backbone for
attachment to another molecule or surface, rather than at the
terminus of the polymer. Although these compounds can be considered
linear, they are often referred to as "branched" and are
distinguished from conventional, linear PEG derivatives since these
molecules typically comprise a pair of mPEG- molecules that have
been joined by their reactive end moieties to another moiety, which
can be represented structurally as -T-, and that includes a
reactive moiety, --Z, extending from the polymer backbone. These
compounds have a general structure that can be represented as
##STR00009##
These monofunctional mPEG polymer derivatives show a branched
structure when linked to another compound. One such branched form
of mPEG with a single active binding site, --Z, has been prepared
by substitution of two of the chloride atoms of
trichloro-s-triazine with mPEG to make mPEG-disubstituted
chlorotriazine. The third chloride is used to bind to protein. An
mPEG disubstituted chlorotriazine and its synthesis are disclosed
in Wada, H., Imamura, I., Sako, M., Katagiri, S., Tarui, S.,
Nishimura, H., and Inada, Y. (1990) Antitumor enzymes: polyethylene
glycol-modified asparaginase. Ann. N. Y. Acad. Sci. 613, 95-108.
Synthesis of mPEG disubstituted chlorotriazine is represented
structurally below.
##STR00010##
However, mPEG-disubstituted chlorotriazine and the procedure used
to prepare it present severe limitations because coupling to
protein is highly nonselective. Several types of amino acids other
than lysine are attacked and many proteins are inactivated. The
intermediate is toxic. Also, the mPEG-disubstituted chlorotriazine
molecule reacts with water, thus substantially precluding
purification of the branched mPEG structure by commonly used
chromatographic techniques in water.
A branched mPEG with a single activation site based on coupling of
mPEG to a substituted benzene ring is disclosed in European Patent
Application Publication No. 473 084 A2. However, this structure
contains a benzene ring that could have toxic effects if the
structure is destroyed in a living organism.
Another branched mPEG with a single activation site has been
prepared through a complex synthesis in which an active succinate
moiety is attached to the mPEG through a weak ester linkage that is
susceptible to hydrolysis. An mPEG-OH is reacted with succinic
anhydride to make the succinate. The reactive succinate is then
activated as the succinimide. The synthesis, starting with the
active succinimide, includes the following steps, represented
structurally below.
##STR00011##
The mPEG activated as the succinimide, mPEG succinimidyl succinate,
is reacted in the first step as shown above with norleucine. The
symbol --R in the synthesis represents the n-butyl moiety of
norleucine. The mPEG and norleucine conjugate (A) is activated as
the succinimide in the second step by reaction with N-hydroxy
succinimide. As represented in the third step, the mPEG and
norleucine conjugate activated as the succinimide (H) is coupled to
the alpha and epsilon amino moieties of lysine to create an mPEG
disubstituted lysine (C) having a reactive carboxyl moiety. In the
fourth step, the mPEG disubstituted lysine is activated as the
succinimide.
The ester linkage formed from the reaction of the mPEG-OH and
succinic anhydride molecules is a weak linkage that is
hydrolytically unstable. In vivo application is therefore limited.
Also, purification of the branched mPEG is precluded by commonly
used chromatographic techniques in water, which normally would
destroy the molecule.
The molecule also has relatively large molecular fragments between
the carboxyl group activated as the succinimide and the mPEG
moieties due to the number of steps in the synthesis and to the
number of compounds used to create the fragments. These molecular
fragments are sometimes referred to as "linkers" or "spacer arms,"
and have the potential to act as antigenic sites promoting the
formation of antibodies upon injection and initiating an
undesirable immunological response in a living organism.
SUMMARY OF THE INVENTION
The invention provides a branched or "multi-armed" amphiphilic
polymer derivative that is monofunctional, hydrolytically stable,
can be prepared in a simple, one-step reaction, and possesses no
aromatic moieties in the linker fragments forming the linkages with
the polymer moieties. The derivative can be prepared without any
toxic linkage or potentially toxic fragments. Relatively pure
polymer molecules of high molecular weight can be created. The
polymer can be purified by chromotography in water. A multi-step
method can be used if it is desired to have polymer arms that
differ in molecular weight. The polymer arms are capped with
relatively nonreactive end groups. The derivative can include a
single reactive site that is located along the polymer backbone
rather than on the terminal portions of the polymer moieties. The
reactive site can be activated for selective reactions.
The multi-armed polymer derivative of the invention having a single
reactive site can be uses' for, among other things, protein
modification with a high retention of protein activity. Protein and
enzyme activity can be preserved and in some cases is enhanced. The
single reactive site can be converted to a functional group for
highly selective coupling to proteins, enzymes, and surfaces. A
larger, more dense polymer cloud can be created surrounding a
biomolecule with fewer attachment points to the biomolecule as
compared to conventional polymer derivatives having terminal
functional groups. Hydrolytically weak ester linkages can be
avoided. Potentially harmful or toxic products of hydrolysis can be
avoided. Large linker fragments can be avoided so as to avoid an
antigenic response in living organisms. Cross linking is
avoided.
The molecules of the invention can be represented structurally as
poly.sub.a-P--CR(-Q-poly.sub.b)-Z or:
##STR00012##
Poly.sub.a and poly.sub.b represent nonpeptidic and substantially
nonreactive water soluble polymeric arms that may be the same or
different. C represents carbon. P and Q represent linkage fragments
that may be the same or different and that join polymer arms
poly.sub.a and poly.sub.b, respectively, to C by hydrolytically
stable linkages in the absence of aromatic rings in the linkage
fragments. R is a moiety selected from the group consisting of H,
substantially nonreactive, usually alkyl, moieties, and linkage
fragments attached by a hydrolytically stable linkage in the
absence of aromatic rings to a nonpeptidic and substantially
nonreactive water soluble polymeric arm. The moiety --Z comprises a
moiety selected from the group consisting of moieties having a
single site reactive toward nucleophilic moieties, sites that can
be converted to sites reactive toward nucleophilic moieties, and
the reaction product of a nucleophilic moiety and moieties having a
single site reactive toward nucleophilic moieties.
Typically, the moiety --P--CR(-Q-)-Z is the reaction product of a
linker moiety and the reactive site of monofunctional, nonpeptidic
polymer derivatives, poly.sub.a-W and poly.sub.b-W, in which W is
the reactive site. Polymer arms poly.sub.a and poly.sub.b are
nonpeptidic polymers and can be selected from polymers that have a
single reactive moiety that can be activated for hydrolytically
stable coupling to a suitable linker moiety. The linker has the
general structure X--CR--(Y)--Z, in which X and Y represent
fragments that contain reactive sites for coupling to the polymer
reactive site W to form linkage fragments P and Q,
respectively.
In one embodiment, at least one of the polymer arms is a
poly(ethylene glycol) moiety capped with an essentially nonreactive
end group, such as a monomethoxy-poly(ethylene glycol) moiety
("mPEG-"), which is capped with a methyl end group, CH.sub.3--. The
other branch can also be an mPEG moiety of the same or different
molecular weight, another poly(ethylene glycol) moiety that is
capped with an essentially nonreactive end group other than methyl,
or a different nonpeptidic polymer moiety that is capped with a
nonreactive end group such as a capped poly(alkylene oxide), a
poly(oxyethylated polyol), a poly(olefinic alcohol), or others.
For example, in one embodiment poly.sub.a and poly.sub.b are each
monomethoxy-poly(ethylene glycol) ("mPEG") of the same or different
molecular weight. The mPEG-disubstituted derivative has the general
structure mPEG.sub.a-P--CH(-Q-mPEG.sub.b)-Z. The moieties
mPEG.sub.a- and mPEG.sub.b- have the structure
CH.sub.3--(CH.sub.4CH.sub.2O).sub.nCH.sub.2CH.sub.2-- and n may be
the same or different for mPEG.sub.a and mPEG.sub.b. Molecules
having values of n of from 1 to about 1,150 are contemplated.
The linker fragments P and Q contain hydrolytically stable linkages
that may be the same or different depending upon the functional
moiety on the mPEG molecules and the molecular structure of the
linker moiety used to join the mPEG moieties in the method for
synthesizing the multi-armed structure. The linker fragments
typically are alkyl fragments containing amino or thiol residues
forming a linkage with the residue of the functional moiety of the
polymer. Depending on the degree of substitution desired, linker
fragments P and Q can include reactive sites for joining additional
monofunctional nonpeptidic polymers to the multi-armed
structure.
The moiety --R can be a hydrogen atom, H, a nonreactive fragment,
or, depending on the degree of substitution desired, R can include
reactive sites for joining additional monofunctional nonpeptidic
polymers to the multi-armed structure.
The moiety --Z can include a reactive moiety for which the
activated nonpeptidic polymers are not selective and that can be
subsequently activated for attachment of the derivative to enzymes,
other proteins, nucleotides, lipids, liposomes, other molecules,
solids, particles, or surfaces. The moiety --Z can include a
linkage fragment --R.sub.2. Depending on the degree of substitution
desired, the R.sub.2 fragment can include reactive sites for
joining additional monofunctional nonpeptidic polymers to the
multi-armed structure.
Typically, the --Z moiety includes terminal functional moieties for
providing linkages to reactive sites on proteins, enzymes,
nucleotides, lipids, liposomes, and other materials. The moiety --Z
is intended to have a broad interpretation and to include the
reactive moiety of monofunctional polymer derivatives of the
invention, activated derivatives, and conjugates of the derivatives
with polypeptides and other substances. The invention includes
biologically active conjugates comprising a biomolecule, which is a
biologically active molecule, such as a protein or enzyme, linked
through an activated moiety to the branched polymer derivative of
the invention. The invention includes biomaterials comprising a
solid such as a surface or particle linked through an activated
moiety to the polymer derivatives of the invention.
In one embodiment, the polymer moiety is an mPEG moiety and the
polymer derivative is a two-armed mPEG derivative based upon
hydrolytically stable coupling of mPEG to lysine. The mPEG moieties
are represented structurally as
CH.sub.3O--(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2-- wherein n
may be the same or different for poly.sub.a- and poly.sub.b- and
can be from 1 to about 1,150 to provide molecular weights of from
about 100 to 100,000. The --R moiety is hydrogen. The --Z moiety is
a reactive carboxyl moiety. The molecule is represented
structurally as follows:
##STR00013##
The reactive carboxyl moiety of hydrolytically stable
mPEG-disubstituted lysine, which can also be called alpha,
epsilon-mPEG lysine, provides a site for interacting with ion
exchange chromatography media and thus provides a mechanism for
purifying the product. These purifiable, high molecular weight,
monofunctional compounds have many uses. For example,
mPEG-disubstituted lysine, activated as succinimidyl ester, reacts
with amino groups in enzymes under mild aqueous conditions that are
compatible with the stability of most enzymes. The
mPEG-disubstituted lysine of the invention, activated as the
succinimidyl ester, is represented as follows:
##STR00014##
The invention includes methods of synthesizing the polymers of the
invention. The methods comprise reacting an active suitable polymer
having the structure poly-W with a linker moiety having the
structure X--CR--(Y)Z to form poly.sub.a-P--CR(-Q-poly.sub.b)-Z.
The poly moiety in the structure poly-W can be either poly.sub.a or
poly.sub.b and is a polymer having a single reactive moiety W. The
W moiety is an active moiety that is linked to the polymer moiety
directly or through a hydrolytically stable linkage. The moieties X
and Y in the structure X--CR--(Y)Z are reactive with w to form the
linkage fragments Q and P, respectively. If the moiety R includes
reactive sites similar to those of X and Y, then R can also be
modified with a poly-W, in which the poly can be the same as or
different from poly.sub.a or poly.sub.b. The moiety Z normally does
not include a site that is reactive with W. However, X, Y, R, and Z
can each include one or more such reactive sites for preparing
monofunctional polymer derivatives having more than two
branches.
The method of the invention typically can be accomplished in one or
two steps. The method can include additional steps for preparing
the compound poly-W and for converting a reactive Z moiety to a
functional group for highly selective reactions.
The active Z moiety includes a reactive moiety that is not reactive
with W and can be activated subsequent to formation of
poly.sub.a-P--CR(-Q-poly.sub.b)-Z for highly selective coupling to
selected reactive moieties of enzymes and ocher proteins or
surfaces or any molecule having a reactive nucleophilic moiety for
which it is desired to modify the characteristics of the
molecule.
In additional embodiments, the invention provides a multi-armed
mPEG derivative for which preparation is simple and
straightforward. Intermediates are water stable and thus can be
carefully purified by standard aqueous chromatographic techniques.
Chlorotriazine activated groups are avoided and more highly
selective functional groups are used for enhanced selectivity of
attachment and much less loss of activity upon coupling of the mPEG
derivatives of the invention to proteins, enzymes, and other
peptides. Large spacer arms between the coupled polymer and protein
are avoided to avoid introducing possible antigenic sites. Toxic
groups, including triazine, are avoided. The polymer backbone
contains no hydrolytically weak ester linkages that could break
down during in vivo applications. Monofunctional polymers of double
the molecular weight as compared to the individual mPEG moieties
can be provided, with mPEG dimer structures having molecular
weights of up to at least about 50,000, thus avoiding the common
problem of difunctional impurities present in conventional, linear
mPEGs.
BRIEF DESCRIPTION OF THE DRAWING
FIGS. 1(a), 1(b), and 1(c) illustrate the time course of digestion
of ribonuclease (.circle-solid.), conventional, linear
mPEG-modified ribonuclease (.largecircle.), and ribonuclease
modified with a multi-armed mPEG of the invention (.box-solid.) as
assessed by enzyme activity upon incubation with pronase (FIG.
1(a)), elastase (FIG. 1(b)), and subtilisin (FIG. 1(c)).
FIGS. 2(a) and 2(b) illustrate stability toward heat (FIG. 2(a))
and pH (FIG. 2(b)) of ribonuclease (.circle-solid.), linear
mPEG-modified ribonuclease (.largecircle.), and ribonuclease
modified with a multi-armed mPEG of the invention (.quadrature.).
FIG. 2(a) is based on data taken after a 15 minute incubation
period at the indicated temperatures. FIG. 2(b) is based on data
taken over a 20 hour period at different pH values.
FIGS. 3(a) and 3(b) illustrate the time course of digestion for
catalase (.circle-solid.), linear mPEG-modified catalase
(.largecircle.), and catalase modified with a multi-armed mPEG of
the invention (.box-solid.) as assessed by enzyme activity upon
incubation with pronase (FIG. 3(a)) and trypsin (FIG. 3(b)).
FIG. 4 illustrates the stability of catalase (.circle-solid.),
linear mPEG-modified catalase (.quadrature.), and catalase modified
with a multi-armed mPEG of the invention (.largecircle.) for 20
hours incubation at the indicated pH values.
FIG. 5 illustrates the time course of digestion of asparaginase
(.circle-solid.), linear mPEG-modified asparaginase
(.largecircle.), and asparaginase modified with a multi-armed mPEG
of the invention (.box-solid.) as assessed by enzyme activity assay
upon trypsin incubation.
FIG. 6 illustrates the time course of autolysis of trypsin
(.circle-solid.), linear mPEG-modified trypsin (.box-solid.), and
trypsin modified with a multi-armed mPEG of the invention
(.tangle-solidup.) evaluated as residual activity towards TAME
(alpha N-p-tosyl-arginine methyl ester).
DETAILED DESCRIPTION
I. Preparation of a Hydrolytically Stable mPEG-Disubstituted
Lysine
Two procedures are describes for the preparation of a
hydrolytically stable, two-armed, mPEG-disubstituted lysine. The
first procedure is a two step procedure, meaning that the lysine is
substituted with each of the two mPEG moieties in separate reaction
steps. Monomethoxy-poly(ethylene glycol) arms of different lengths
or of the same length can be substituted onto the lysine molecule,
if desired, using the two step procedure. The second procedure is a
one step procedure in which the lysine molecule is substituted with
each of the two mPEG moieties in a single reaction step. The one
step procedure is suitable for preparing mPEG-disubstituted lysine
having mPEG moieties of the same length.
Unlike prior multisubstituted structures, no aromatic ring is
present in the linkage joining the nonpeptidic polymer arms
produced by either the one or two step methods described below that
could result in toxicity if the molecule breaks down in vivo. No
hydrolytically weak ester linkages are present in the linkage.
Lengthy linkage chains that could promote an antigenic response are
avoided.
The terms "group," "functional group," "moiety," "active moiety,"
"reactive site," "radical," and similar terms are somewhat
synonymous in the chemical arts and are used in the art and herein
to refer to distinct, definable portions or units of a molecule or
fragment of a molecule. "Reactive site," "functional group," and
"active moiety" refer to units that perform some function or have a
chemical activity and are reactive with other molecules or portions
of molecules. In this sense a protein or a protein residue can be
considered as a molecule and as a functional moiety when coupled to
a polymer. A polymer, such as mPEG-COOH has a reactive site, the
carboxyl moiety, --COOH, that can be converted to a functional
group for selective reactions and attachment to proteins and linker
moieties. The converted polymer is said to be activated and to have
an active moiety, while the --COOH group is relatively nonreactive
in comparison to an active moiety.
The term "nonreactive" is used herein primarily to refer to a
moiety that does not readily react chemically with other moieties,
such as the methyl alkyl moiety. However, the term "nonreactive"
should be understood to exclude carboxyl and hydroxyl moieties,
which, although relatively nonreactive, can be converted to
functional groups that are of selective reactivity.
The term "biologically active" means a substance, such as a
protein, lipid, or nucleotide that has some activity or function in
a living organism or in a substance taken from a living organism.
For example, an enzyme can catalyze chemical reactions. The term
"biomaterial" is somewhat imprecise, and is used herein to refer to
a solid material or particle or surface that is compatible with
living organisms or tissue or fluids. For example, surfaces that
contact blood, whether in vitro or in vivo, can be made nonfouling
by attachment of the polymer derivatives of the invention so that
proteins do not become attached to the surface.
A. Two Step Procedure
For the two step procedure, an activated mPEG is prepared for
coupling to free lysine monomer and then the lysine monomer is
disubstituted with the activated mPEG in two steps. The first step
occurs in aqueous buffer. The second step occurs in dry methylene
chloride. The active moiety of the mPEG for coupling to the lysine
monomer can be selected from a number of activating moieties having
leaving moieties that are reactive with the amino moieties of
lysine monomer. A commercially available activated mPEG,
mPEG-p-nitrophenylcarbonate, the preparation of which is discussed
below was used to exemplify the two step procedure.
The two step procedure can be represented structurally as
follows:
##STR00015##
Step 1. Preparation of mPEG-monosubstituted lysine. Modification of
a single lysine amino group was accomplished with
mPEG-p-nitrophenylcarbonate in aqueous solution where both lysine
and mPEG-p-nitrophenylcarbonate are soluble. The
mPEG-p-nitrophenylcarbonate has only limited stability in aqueous
solution. However, lysine is not soluble in organic solvents in
which the activated mPEG is stable. Consequently, only one lysine
amino group is modified by this procedure. NMR confirms that the
epsilon amino group is modified. Nevertheless, the procedure allows
ready chloroform extraction of mPEG-monosubstituted lysine from
unreacted lysine and other water soluble by-products, and so the
procedure provides a desirable monosubstituted product for
disubstitution.
To prepare the mPEG-monosubstituted lysine, 353 milligrams of
lysine, which is about 2.5 millimoles, was dissolved in 20
milliliters of water at a pH of about 8.0 to 8.3. Five grams of
mPEG-p-nitrophenylcarbonate of molecular weight 5,000, which is
about 1 millimole, was added in portions over 3 hours. The pH was
maintained at 8.3 with 0.2 N NaOH. The reaction mixture was stirred
overnight at room temperature. Thereafter, the reaction mixture was
cooled to 0.degree. C. and brought to a pH of about 3 with 2 N HCl.
Impurities were extracted with diethyl ether. The mPEG
monosubstituted lysine, having the mPEG substituted at the epsilon
amino group of lysine as confirmed by NMR analysis, was extracted
three times with chloroform. The solution was dried. After
concentration, the solution was added drop by drop to diethyl ether
to form a precipitate. The precipitate was collected and then
crystallized from absolute ethanol. The percentage of modified
amino groups was 53%, calculated by colorimetric analysis.
Step 2. Preparation of mPEG-Disubstituted Lysing. The
mPEG-monosubstituted lysine product from step 1 above is soluble in
organic solvents and so modification of the second lysine amino
moiety can be achieved by reaction in dry methylene chloride.
Activated mPEG, mPEG-p-nitrophenylcarbonate, is soluble and stable
in organic solvents and can be used to modify the second lysine
amino moiety.
Triethylamine ("TEA") was added to 4.5 grams of
mPEG-monosubstituted lysine, which is about 0.86 millimoles. The
mixture of TEA and mPEG-monosubstituted lysine was dissolved in 10
milliliters of anhydrous methylene chloride to reach a pH of 8.0.
Four and nine tenths grams of mPEG-p-nitrophenycarbonate of
molecular weight 5,000, which is 1.056 millimoles, was added over 3
hours to the solution. If it is desirable to make an mPEG
disubstituted compound having mPEG arms of different lengths, then
a different molecular weight mPEG could have been used. The pH was
maintained at 8.0 with TEA. The reaction mixture was refluxed for
72 hours, brought to room temperature, concentrated, filtered,
precipitated with diethyl ether and then crystallized in a minimum
amount of hot ethanol. The excess of activated mPEG,
mPEG-p-nitrophenycarbonate, was deactivated by hydrolysis in an
alkaline aqueous medium by stirring overnight at room temperature.
The solution was cooled to 0.degree. C. and brought to a pH of
about 3 with 2 N HCl.
p-Nitrophenol was removed by extraction with diethyl ether.
Monomethyl-poly(ethylene glycol)-disubstituted lysine and remaining
traces of mPEG were extracted from the mixture three times with
chloroform, dried, concentrated, precipitated with diethyl ether
and crystallized from ethanol. No unreacted lysine amino groups
remained in the polymer mixture as assessed by colorimetric
analysis.
Purification of mPEG-disubstituted lysine and removal of mPEG were
accomplished by gel filtration chromatography using a Bio Gel P100
(Bio-Rad) column. The column measured 5 centimeters by 50
centimeters. The eluent was water. Fractions of 10 milliliters were
collected. Up to 200 milligrams of material could be purified for
each run. The fractions corresponding to mPEG-disubstituted lysine
were revealed by iodine reaction. These fractions were pooled,
concentrated, and then dissolved in ethanol and concentrated. The
mPEG-disubstituted lysine product was dissolved in methylene
chloride, precipitated with diethyl ether, and crystallized from
ethanol.
The mPEG-disubstituted lysine was also separated from unmodified
mPEG-OH and purified by an alternative method. Ion exchange
chromatography was performed on a QAE Sephadex A50 column
(Pharmacia) that measured 5 centimeters by 80 centimeters. An 8.3
mM borate buffer of pH 8.9 was used. This alternative procedure
permitted fractionation of a greater amount of material per run
than the other method above described (up to four grams for each
run).
For both methods of purification, purified mPEG-disubstituted
lysine of molesular weight 10,000, titrated with NaOH, showed that
100% of the carboxyl groups were free carboxyl groups. These
results indicate chat the reaction was complete and the product
pure.
The purified mPEG-disubstituted lysine was also characterized by
.sup.1H-NMR on a 200 MHz Bruker instrument in dimethyl sulfoxide,
d6 at a 5% weight to volume concentration. The data confirmed the
expected molecular weight of 10,000 for the polymer. The chemical
shifts and assignments of the protons in the mPEG-disubstituted
lysine are as follows: 1.2-1.4 ppm (multiplet, 6H, methylenes 3,4,5
of lysine); 1.6 ppm (multiplet, 2H, methylene 6 of lysine); 3.14
ppm (s, 3H, terminal mPEG methoxy); 3.49 ppm (s, mPEG backbone
methylene); 4.05 ppm (t, 2H, --CH.sub.2, --OCO--); 7.18 ppm (t, 1H,
--NH-- lysine); and 7.49 ppm (d, 1H, --NH-- lysine).
The above signals are consistent with the reported structure since
two different carbamate NH protons are present. The first carbamate
NH proton (at 7.18 ppm) shows a triplet for coupling with the
adjacent methylene group. The second carbamate NH proton (at 7.49
ppm) shows a doublet because of coupling with the .alpha.-CH of
lysine. The intensity of these signals relative to the mPEG
methylene peak is consistent with the 1:1 ratio between the two
amide groups and the expected molecular weight of 10,000 for the
polymer.
The two step procedure described above allows polymers of different
types and different lengths to be linked with a single reactive
site between them. The polymer can be designed to provide a polymer
cloud of custom shape for a particular application.
The commercially available activated mPEG,
mPEG-p-nitrophenylcarbonate, is available from Shearwater Polymers,
Inc. in Huntsville, Ala. This compound was prepared by the
following procedure, which can be represented structurally as
follows:
##STR00016##
Five grams of mPEG-OH of molecular weight 5,000, or 1 millimole,
were dissolved in 120 milliliters of toluene and dried
azeotropically for 3 hours. The solution was cooled to room
temperature and concentrated under vacuum. Reactants added to the
concentrated solution under stirring at 0.degree. C. were 20
milliliters of anhydrous methylene chloride and 0.4 g of
p-nitrophenylchloroformate, which is 2 millimoles. The pH of the
reaction mixture was maintained at 8 by adding. 0.28 milliliters of
triethylamine ("TEA"), which is 2 millimoles. The reaction mixture
was allowed to stand overnight at room temperature. Thereafter, the
reaction mixture was concentrated under vacuum to about 10
milliliters, filtered, and dropped into 100 milliliters of stirred
diethyl ether. A precipitate was collected from the diethyl ether
by filtration and crystallized twice from ethyl acetate. Activation
of mPEG was determined to be 98%. Activation was calculated
spectrophotometrically on the basis of the absorption at 400 nm in
alkaline media after 15 minutes of released 4-nitrophenol
(.epsilon. of p-nitrophenol at 400 nm equals 17,000).
B. One Step Procedure
In the one step procedure, mPEG disubstituted lysine is prepared
from lysine and an activated mPEG in a single step as represented
structurally below:
##STR00017##
Except for molecular weight attributable to a longer PEG backbone
in the activated mPEG used in the steps below, the mPEG
disubstituted lysine of the one step procedure does not differ
structurally from the mPEG disubstituted lysine of the two step
procedure. It should be recognized that the identical compound,
having the same molecular weight, can be prepared by either
method.
Preparation of mPEG disubstituted lysine by the one step procedure
proceeded as follows: Succinimidylcarbonate mPEG of molecular
weight about 20,000 was added in an amount of 10.8 grams, which is
5.4.times.10.sup.-4 moles, to 40 milliliters of lysine HCl
solution. The lysine HCL solution was in a borate buffer of pH 8.0.
The concentration was 0.826 milligrams succinimidylcarbonate mPEG
per milliliter of lysine HCL solution, which is
1.76.times.10.sup.-4 moles. Twenty milliliters of the same buffer
was added. The solution pH was maintained at 8.0 with aqueous NaOH
solution for the following 8 hours. The reaction mixture was
stirred at room temperature for 24 hours.
Thereafter, the solution was diluted with 300 milliliters of
deionized water. The pH of the solution was adjusted to 3.0 by the
addition of oxalic acid. The solution was then extracted three
times with dichloromethane. The combined dichloromethane extracts
ware dried with anhydrous sodium sulphate and filtered. The
filtrate was concentrated to about 30 milliliters. The product, an
impure mPEG disubstituted lysine, was precipitated with about 200
milliliters of cold ethyl ether. The yield was 90%.
Nine grams of the above impure mPEG-disubstituted lysine reaction
product was dissolved in 4 liters of distilled water and then
loaded onto a column of DEAF Sepharose FF, which is 500 milliliters
of gel equilibrated with 1500 milliliters of boric acid in a 0.5%
sodium hydroxide buffer at a pH of 7.0. The loaded system was then
washed with water. Impurities of succinimidylcarbonate mPEG and
mPEG-monosubstituted lysine, both of molecular weight about 20,000,
were washed off the column. However, the desired mPEG disubstituted
lysine of molecular weight 20,000 was eluted with 10 mM NaCl. The
pH of the eluate was adjusted to 3.0 with oxalic acid and then mPEG
disubstituted lysine was extracted with dichloromethane, dried with
sodium sulphate, concentrated, and precipitated with ethyl ether.
Five and one tenth grams of purified mPEG disubstituted lysine were
obtained. The molecular weight was determined to be 38,000 by gel
filtration chromatography and 36,700 by potentiometric
titration.
The one step procedure is simple in application and is useful for
producing high molecular weight dimers that have polymers of the
same type and length linked with a single reactive site between
them.
Additional steps are represented below for preparing
succinimidylcarbonate mPEG for disubstitution of lysine.
##STR00018##
Succinimidylcarbonate mPEG was prepared by dissolving 30 grams of
mPEG-OH of molecular weight 20,000, which is about 1.5 millimoles,
in 120 milliliters of toluene. The solution was dried
azeotropically for 3 hours. The dried solution was cooled to room
temperature. Added to the cooled and dried solution were 20
milliliters of anhydrous dichloromethane and 2.33 milliliters of a
20% solution of phosgene in toluene. The solution was stirred
continuously for a minimum of 16 hours under a hood due to the
highly toxic fumes.
After distillation of excess phosgene and solvent, the remaining
syrup, which contained mPEG chlorocarbonate, was dissolved in 100
milliliters of anhydrous dichloromethane, as represented above. To
this solution was added 3 millimoles of triethylamine and 3
millimoles of N-hydroxysuccinimide. The reaction mixture remained
standing at room temperature for 24 hours. Thereafter, the solution
was filtered through a silica gel bed of pore size 60 Angstroms
that had been wetted with dichloromethane. The filtrate was
concentrated to 70 milliliters. Succinimidylcarbonate mPEG of
molecular weight about 20,000 was precipitated in ether and dried
in vacuum for a minimum or b hours. The yield was 90%.
Succinimidylcarbonate-mPEG is available commercially from
Shearwater Polymers in Huntsville, Ala.
The mPEG disubstituted lysine of the invention can be represented
structurally more generally as poly, --P--CR(-Q-poly.sub.b)-Z
or:
##STR00019##
For the mPEG disubstituted lysines described above, --P--CR(-Q-)-Z
is the reaction product of a precursor linker moiety having two
reactive amino groups and active monofunctional precursors of poly,
and poly, that have been joined to the linker moiety at the
reactive amino sites. Linker fragments Q and P contain carbamate
linkages formed by joining the amino containing portions of the
lysine molecule with the functional group with which the mPEG was
substituted. The linker fragments are selected from
--O--C(O)NH(CH.sub.2).sub.4-- and --O--C(O)NH-- and are different
in the exemplified polymer derivative. However, it should be
recognized that P and Q could both be --O--C(O)NH(CH.sub.2).sub.4--
or --O--C(O)NH-- or some other linkage fragment, as discussed
below. The moiety represented by R is hydrogen, H. The moiety
represented by Z is the carboxyl group, COOH. The moieties P, R, Q,
and Z are all joined to a central carbon atom.
The nonpeptidic polymer arms, poly.sub.a and poly.sub.b, are mPEG
moieties mPEG.sub.a and mPEG.sub.b, respectively, and are the same
on each of the linker fragments Q and P for the examples above. The
mPEG moieties have a structure represented as
CH.sub.3O--(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2--. For the
mPEG disubstituted lysine made by the one step method, n is about
454 to provide a molecular weight for each mPEG moiety of 20,000
and a dimer molecular weight of 40,000. For the mPEG disubstituted
lysine made by the two step method, n is about 114 to provide a
molecular weight for each mPEG moiety of 5,000 and a dimer
molecular weight of 10,000.
Lysine disubstituted with mPEG and having as dimer molecular
weights of 10,000 and 40,060 and procedures for preparation of
mPEG-disubstituted lysine have been shown. However, it should be
recognized that mPEG disubstituted lysine and other multi-armed
compounds of the invention can be made in a variety of molecular
weights, including ultra high molecular weights. High molecular
weight monofunctional PEGs are otherwise difficult to obtain.
Polymerization of ethylene oxide to yield mPEGs usually produces
molecular weights of up to about 20,000 to 25,000 g/mol.
Accordingly, two-armed mPEG disubstituted lysines of molecular
weight of about 40,000 to 50,000 can be made according to the
invention. Higher molecular weight lysine disubstituted PEGS can be
made if the chain length of the linear mPEGs is increased, up to
about 100,000. Higher molecular weights can also be obtained by
adding additional monofunctional nonpeptidic polymer arms to
additional reactive sites on a linker moiety, within practical
limits of steric hindrance. However, no unreacted active sites on
the linker should remain that could interfere with the
monofunctionality of the multi-armed derivative. Lower molecular
weight disubstituted mPEGs can also be made, if desired, down to a
molecular weight of about 100 to 200.
It should be recognized that a wide variety of linker fragments P
and Q are available, although not necessarily with equivalent
results, depending on the precursor linker moiety and the
functional moiety with which the activated mPEG or other
nonpeptidic monofunctional polymer is substituted and tram which
the linker fragments result. Typically, the linker fragments will
contain the reaction products of portions of linker moieties that
have reactive amino and/or thiol moieties and suitably activated
nonpeptidic, monofunctional, water soluble polymers.
For example, a wide variety of activated mPEGs are available that
form a wide variety of hydrolytically stable linkages with reactive
amino moieties. Linkages can be selected from the group consisting
of amide, amine, ether, carbamate, which are also called urethane
linkages, urea, thiourea, thiocarbamate, thiocarbonate, thioether,
thioester, dithiocarbonate linkages, and others. However,
hydrolytically weak ester linkages and potentially toxic aromatic
moieties are to be avoided.
Hydrolytic stability of the linkages means that the linkages
between the polymer arms and the linker moiety are stable in water
and that the linkages do not react with water at useful pHs for an
extended period of time of at least several days, and potentially
indefinitely. Most proteins could be expected to lose their
activity at a caustic pH of 11 or higher, so the derivatives should
be stable at a pH of less than about 11.
Examples of the above linkages and their formation from activated
mPEG and lysine are represented structurally below.
a) Formation of Amide Linkage
##STR00020## ##STR00021##
b) Formation of Carbamate Linkage
##STR00022##
c) Formation of Urea Linkage
##STR00023##
d) Formation of Thiourea Linkage
##STR00024##
e) Formation of Amine Linkage
##STR00025##
One or both of the reactive amino moieties, --NH.sub.2, of lysine
or another linker moiety can be replaced with thiol moieties, --SH.
Where the linker moiety has a reactive thiol moiety instead of an
amino moiety, then the linkages can be selected from the group
consisting of thioester, thiocarbonate, thiocarbamate,
dithiocarbamate, thioether linkages, and others. The above linkages
and their formation from activated mPEG and lysine in which both
amino moieties have been replaced with thiol moieties are
represented structurally below.
a) Formation of Thioester Linkage
##STR00026##
b) Formation of Thiocarbonate Linkage
##STR00027##
c) Formation of Thiocarbamate Linkage
##STR00028##
d) Formation of Dithiocarbamate Linkage
##STR00029##
e) Formation of Thioether Linkage
##STR00030##
It should be apparent that the mPEG or other monofunctional polymer
reactants can be prepared with a reactive amino moiety and then
linked to a suitable linker moiety having reactive groups such as
those shown above on the mPEG molecule to form hydrolytically
stable linkages as discussed above. For example, the amine linkage
could be formed as follows:
##STR00031##
Examples of various active electrophilic moieties useful for
activating polymers or linking moieties for biological and
biotechnical applications in which the active moiety is reacted to
form hydrolytically stable linkages in the absence of arcmatic
moieties include trifluoromethylsulfonate, isocyanate,
isothiocyanate, active esters, active carbonates, various
aldehydes, various sulfones, including chloroethylsulfone and
vinylsulfone, maleimide, iodoacetamide, and iminoesters. Active
esters include N-hydroxylsuccinimidyl ester. Active carbonates
include N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate,
and trichlorophenylcarbonate. These electrophilic moieties are
examples of those that are suitable as Ws in the structure poly-W
and as Xs and Ys in the linker structure X--CR(--Y)--Z.
Nucleophilic moieties for forming the linkages can be amino, thiol,
and hydroxyl. Hydroxyl moieties form hydrolytically stable linkages
with isocyanate electrophilic moieties. Also, it should be
recognized that the linker can be substituted with different
nucleophilic or electrophilic moieties or both electrophilic and
nucleophilic moieties depending on the active moieties on the
monofunctional polymers with which the linker moiety is to be
substituted.
Linker moieties other than lysine are available for activation and
for disubstitution or multisubstitution with mPEG and related
polymers for creating multi-armed structures in the absence of
aromatic moieties in the structure and that are hydrolytically
stable. Examples of such linker moieties include chose having more
than one reactive site for attachment of various monofunctional
polymers.
Linker moieties can be synthesized to include multiple reactive
sites such as amino, thiol, or hydroxyl groups for joining multiple
suitably activated mPEGs or other nonpeptidic polymers to the
molecule by hydrolytically stable linkages, if it is desired to
design a molecule having multiple nonpeptidic polymer branches
extending from one or more of the linker arm fragments. The linker
moieties should also include a reactive site, such as a carboxyl or
alcohol moiety, represented as --Z in the general structure above,
for which the activated polymers are not selective and that can be
subsequently activated for selective reactions for joining to
enzymes, other proteins, surfaces, and the like.
For example, one suitable linker moiety is a diamino alcohol having
the structure
##STR00032##
The diamino alcohol can be disubstituted with activated mPEG or
other suitable activated polymers similar to disubstitution of
lysine and then the hydroxyl moiety can be activated as
follows:
##STR00033##
Other diamino alcohols and alcohols having more than two amino or
other reactive groups for polymer attachment are useful. A suitably
activated mPEG or other monofunctional, nonpeptidic, water soluble
polymer can be attached to the amino groups on such a diamino
alcohol similar to the method by which the same polymers are
attached to lysine as shown above. Similarly, the amino groups can
be replaced with thiol or other active groups as discussed above.
However, only one hydroxyl group, which is relatively nonreactive,
should be present in the --Z moiety, and can be activated
subsequent to polymer substitution.
The moiety --Z can include a reactive moiety or functional group,
which normally is a carboxyl moiety, hydroxyl moiety, or activated
carboxyl or hydroxyl moiety. The carboxyl and hydroxyl moieties are
somewhat nonreactive as compared to the thiol, amino, and other
moieties discussed above. The carboxyl and hydroxyl moieties
typically remain intact when the polymer arms are attached to the
linker moiety and can be subsequently activated. The carboxyl and
hydroxyl moieties also provide a mechanism for purification of the
multisubstituted linker moiety. The carboxyl and hydroxyl moieties
provide a site for interacting with ion exchange chromatography
media.
The moiety --Z may also include a linkage fragment, represented as
R.sub.z in the moiety, which can be substituted or unsubstituted,
branched or linear, and joins the reactive moiety to the central
carbon. Where a reactive group of the --Z moiety is carboxyl, for
activation after substitution with nonpeptidic polymers, then the
--Z moiety has the structure --R.sub.z--COOH if the R, fragment is
present. For hydroxyl, the structure is --R.sub.z--OH. For example,
in the diamino alcohol structure discussed above, R.sub.z is
CH.sub.2. It should be understood that the carboxyl and hydroxyl
moieties normally will extend from the R.sub.z terminus, but need
not necessarily do so.
R.sub.z can also include the reaction product of one or more
reactive moieties including reactive amino, thiol, or other
moieties, and a suitably activated mPEG arm or related nonpeptidic
polymer arm. In the latter event, R.sub.z can have the structure
(-L-poly)-COOH or (-L-poly.sub.c)-OH in which -L- is the reaction
product of a portion of the linker moiety and a suitably activated
nonpeptidic polymer, poly.sub.c-W, which is selected from the same
group as poly.sub.a-W and poly.sub.b-W but can be the same or
different from poly.sub.a-W and poly.sub.b-W.
It is intended that --Z have a broad definition. The moiety --Z is
intended to represent not only the reactive site of the
multisubstituted polymeric derivative that subsequently can be
converted to an active form and its attachment to the central
carbon, but the activated reactive site and also the conjugation of
the precursor activated site with another molecule, whether that
molecule be an enzyme, other protein or polypeptide, a
phospholipid, a preformed liposome, or on a surface to which the
polymer derivative is attached.
The skilled artisan should recognize that Z encompasses the
currently known activating moieties in PEG chemistry and their
conjugates. It should also be recognized that, although the linker
fragments represented by Q and P and R.sub.z should not contain
aromatic rings or hydrolytically weak linkages such as ester
linkages, such rings and such hydrolytically weak linkages may be
present in the active site moiety of --Z or in a molecule joined to
such active site. It may be desirable in some instances to provide
a linkage between, for example, a protein or enzyme and a
multisubstituted polymer derivative that has limited stability in
water. Some amino acids contain aromatic moieties, and it is
intended that the structure Z include conjugates of
multisubstituted monofunctional polymer derivatives with such
molecules or portions of molecules. Activated Zs and Zs including
attached proteins and other moieties are discussed below.
When lysine, the diamino alcohol shown above, or many other
compounds are linkers, then the central carbon has a nonreactive
hydrogen, H, attached thereto. In the general structure
poly.sub.a-P--CR(-Q-poly.sub.b)-Z, R is H. It should be recognized
that the moiety R can be designed to have another substantially
nonreactive moiety, such as a nonreactive methyl or other alkyl
group, or can be the reaction product of one or more reactive
moieties including reactive amino, thiol, or other moieties, and a
suitably activated mPEG arm or related nonpeptidic polymer arm. In
the latter event, R can have the structure -M-poly.sub.d, in which
-M- is the reaction product of a portion of the linker moiety and a
suitably activated nonpeptidic polymer, poly.sub.d-W, which is
selected from the same group as poly.sub.a-W and poly.sub.b-W but
can be the same or different from poly.sub.a-W and
poly.sub.b-W.
For example, multi-armed structures can be made having one or more
mPEGs or other nonpeptidic polymer arms extending from each portion
P, Q, R, and all of which portions extend from a central carbon
atom, C, which multi-armed structures have a single reactive site
for subsequent activation included in the structure represented by
Z. Upon at least the linker fragments P and Q are located at least
one active site for which the monofunctional, nonpeptidic polymers
are selective. These active sites include amino moieties, thiol
moieties, and other moieties as described above.
The nonpeptidic polymer arms tend to mask antigenic properties of
the linker fragment, if any. A linker fragment length of from 1 to
10 carbon atoms or the equivalent has been determined to be useful
to avoid a length that could provide an antigenic site. Also, for
all the linker fragments P, Q, R, and R, there should be an absence
of aromatic moieties in the structure and the linkages should be
hydrolytically stable.
Poly(ethylene glycol) is useful in the practice of the invention
for the nonpeptidic polymer arms attached to the linker fragments.
PEG is used in biological applications because it has properties
that are highly desirable and is generally approved for biological
or biotechnical applications. PEG typically is clear, colorless,
odorless, soluble in water, stable to heat, inert to many chemical
agents, does not hydrolyze or deteriorate, and is nontoxic.
Poly(ethylene glycol) is considered to be biocompatible, which is
to say that PEG is capable of coexistence with living tissues or
organisms without causing harm. More specifically, PEG is not
immunogenic, which is to say that PEG does not tend to produce an
immune response in the body. When attached to a moiety having some
desirable function in the body, the PEG tends to mask the moiety
and can reduce or eliminate any immune response so that an organism
can tolerate the presence of the moiety. Accordingly, the activated
PEGS of the invention should be substantially non-toxic and should
not tend substantially to produce an immune response or cause
clotting or other undesirable effects.
The term "PEG" is used in the art and herein to describe any of
several condensation polymers of ethylene glycol having the general
formula represented by the structure
HO--(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2--OH or, more simply,
as HO-PEG-OH. PEG is also known as polyoxyethylene, polyethylene
oxide, polyglycol, and polyether glycol. PEG can be prepared as
copolymers of ethylene oxide and many other monomers.
Other water soluble polymers than PEG are suitable for similar
modification to create multi-armed structures that can be activated
for selective reactions. These other polymers include poly(vinyl
alcohol) ("PVA"); other poly(alkylene oxides) such as
poly(propylene glycol) ("PPG") and the like; and poly(oxyethylated
polyols) such as poly(oxyethylated glycerol), poly(oxyethylated
sorbitol), and poly(oxyethylated glucose), and the like. The
polymers can be homopolymers or random or block copolymers and
terpolymers based on the monomers of the above polymers, straight
chain or branched, or substituted or unsubstituted similar to mPEG
and other capped, monofunctional PEGs having a single active site
available for attachment to a linker.
Specific examples of suitable additional polymers include
poly(oxazoline), poly(acryloylmorpholine) ("PAcM"), and
poly(vinylpyrrolidone)("PVP"). PVP and poly(oxazoline) are well
known polymers in the art and their preparation and use in the
syntheses described above for mPEG should be readily apparent to
the skilled artisan.
An example of the synthesis of a PVP disubstituted lysine having a
single carboxyl moiety available for activation is shown below. The
disubstituted compound can be purified, activated, and used in
various reactions for modification of molecules and surfaces
similarly to the mPEG-disubstituted lysine described above.
##STR00034##
Poly(acryloylmorpholine) "(PAcM)" functionalized at one end is a
new polymer, the structure, preparation, and characteristics of
which are described in Italian Patent Application No. MI 92 A
0002616, which was published May 17, 1994 and is entitled, in
English, "Polymers Of N-Acryloylmorpholine Functionalized At One
End And Conjugates With Bioactive Materials And Surfaces." Dimer
polymers of molecular weight up to at least about 80,000 can be
prepared using this polymer. The contents of the Italian patent
application are incorporated herein by reference.
PAcM can be used similarly to mPEG or PVP to create multi-armed
structures and ultra-high molecular weight polymers. An example of
a PAcM-disubstituted lysine having a single carboxyl moiety
available for activation is shown below. The disubstituted compound
can be purified, activated, and used in various reactions for
modification of molecules and surfaces similarly to the mPEG- and
PVP-disubstituted lysines described above.
##STR00035##
It should also be recognized that the multi-armed monofunctional
polymers of the invention can be used for attachment to a linker
moiety to create a highly branched monofunctional structure, within
the practical limits of steric hindrance.
II. Activation of mPEG-Disubstituted Lysine and Modification of
Protein Amino Groups.
Schemes are represented below for activating the mPEG-disubstituted
lysine product made by either the one step or two step procedures
and for linking the activated mPEG-disubstituted lysine through a
stable carbamate linkage to protein amino groups to prepare polymer
and protein conjugates. Various other multisubstituted polymer
derivatives as discussed above can be activated similarly.
A. Activation of mPEG Disubstituted Lysine.
Purified mPEG-disubstituted lysine produced in accordance with the
two step procedure discussed above was activated with
N-hydroxysuccinimide to produce mPEG-disubstituted lysine activated
as the succinimidyl ester. The reaction is represented structurally
below:
##STR00036##
Six and two tenths grams of mPEG-disubstituted lysine of molecular
weight 10,000, which is about 0.6 millimoles, was dissolved in 10
milliliters of anhydrous methylene chloride and cooled to 0.degree.
C. N-hydroxysuccinimide and N,N-dicyclohexylcarbodiimide ("DCC")
were added under stirring in the amounts, respectively, of 0.138
milligrams, which is about 1.2 millimoles, and 0.48 milligrams,
which is about 1.2 millimoles. The reaction mixture was stirred
overnight at room temperature. Precipitated dicyclohexylurea was
removed by filtration and the solution was concentrated and
precipitated with diethyl ether. The product, mPEG disubstituted
lysine activated as the succinimidyal ester was crystallized from
ethyl acetate. The yield of esterification, calculated on the basis
of hydroxysuccinimide absorption at 260 nm (produced by
hydrolysis), was over 97% (.epsilon. of hydroxysuccinimide at 260
nm=8,000 m.sup.-1cm.sup.-1). The NMR spectrum was identical to that
of the unactivated carboxylic acid except for the new succinimide
singlet at 2.80 ppm (2Hs)
The procedure previously described for the activation of the
mPEG-disubstituted lysine of molecular weight 10,000 was also
followed for the activation of the higher molecular weight polymer
of molecular weight approximately 40,000 that was produced in
accordance with the one step procedure discussed above. The yield
was over 95% of high molecular weight mPEG-disubstituted lysine
activated as the succinimidyal ester.
It should be recognized that a number of activating groups can be
used to activate the multisubstituted polymer derivatives for
attachment to surfaces and molecules. Any of the activating groups
of the known derivatives of PEG can be applied to the
multisubstituted structure. For example, the mPEG-disubstituted
lysine of the invention was functionalized by activation as the
succinimidyl ester, which can be attached to protein amino groups.
However, there are a wide variety of functional moieties available
for activation of carboxilic acid polymer moieties for attachment
to various surfaces and molecules. Examples of active moieties used
for biological and biotechnical applications include
trifluoroethylsulfonate, isocyanate, isosthiocyanate, active
esters, active carbonates, various aldehydes, various sulfones,
including chloroethylsulfone and vinylsulfone, maleimide,
iodoacetamide, and iminoesters. Active esters include
N-hydroxylsuccinimidyl eater. Active carbonates include
N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate, and
trichlorophenylcarbonate.
A highly useful, new activating group that can be used for highly
selective coupling with thiol moieties instead of amino moieties on
molecules and surfaces is the vinyl sulfone moiety described in
co-pending U.S. patent application Ser. No. 08/151,481, which was
filed on Nov. 12, 1993. the contents of which are incorporated
herein by reference. Various sulfone moieties can be used to
activate a multi-armed structure in accordance with the invention
for thiol selective coupling.
Various examples of activation of --Z reactive moieties to created
--Z activated moieties are presented as follows:
##STR00037##
It should also be recognized that, although the linker fragments
represented by Q and P should not contain aromatic rings or
hydrolytically weak linkages such as ester linkages, such rings and
such hydrolytically weak linkages may be present in the moiety
represented by --Z. It may be desirable in some instances to
provide a linkage between, for example, a protein or enzyme and a
multisubstituted polymer derivative that has limited stability in
water. Some amino acids contain aromatic moieties, and it is
intended that the structure --Z include conjugates of
multisubstituted monofunctional polymer derivatives with such
molecules or portions of molecules.
B. Enzyme Modification
Enzymes were modified with activated, two-armed, mPEG-disubstituted
lysine of the invention of molecular weight about 10,000 that had
been prepared according to the two step procedure and activated as
the succinimidyl ester as discussed above. The reaction is
represented structurally below;
##STR00038##
For comparison, enzymes were also modified with activated,
conventional, linear mPEG of molecular weight 5,000, which was mPEG
with a norleucine amino acid spacer arm activated as the
succinimide. In the discussion of enzyme modification below,
conventional, linear mPEG derivatives with which enzymes are
modified are referred to as "linear mPEG." The activated,
two-armed, mPEG-disubstituted lysine of the invention is referred
to as "two-armed mPEG." Different procedures were used for enzyme
modification depending upon the type of enzyme and the polymer used
so that a similar extent of amino group modification or attachment
for each enzyme could be obtained. Generally, higher molar ratios
of the two-armed mPEG were used. However, in all examples the
enzymes were dissolved in a 0.2 M borate buffer of pH 8.5 to
dissolve proteins. The polymers were added in small portions for
about 10 minutes and stirred for over 1 hour. The amount of polymer
used for modification was calculated on the basis of available
amino groups in the enzyme.
Ribonuclease in a concentration of 1.5 milligrams per milliliter of
buffer was modified at room temperature. Linear and two-armed mPEGs
as described were added at a molar ratio of polymer to protein
amino groups of 2.5:1 and 5:1, respectively. Ribonuclease has a
molecular weight of 13,700 D and 11 available amino groups.
Catalase has a molecular weight of 250,000 D with 112 available
amino groups. Trypsin has a molecular weight of 23,000 D with 16
available amino groups. Erwinia Caratimora asparaginase has a
molecular weight of 141,000 D and 92 free amino groups.
Catalase in a concentration of 2.5 milligrams per milliliter of
buffer was modified at room temperature. Linear and two-armed mPEGs
as described were added at a molar ratio of polymer to protein
amino groups of 5:1 and 10:1, respectively.
Trypsin in a concentration of 4 milligrams per milliliter of buffer
was modified at 0.degree. C. Linear and two-armed mPEGs as
described were added at a molar ratio of polymer to protein amino
groups of 2.5:1.
Asparaginase in a concentration of 6 milligrams per milliliter of
buffer was modified with linear mPEG at room temperature. Linear
mPEG as described was added at a molar ratio of polymer to protein
amino groups of 3:1. Asparaginase in a concentration of 6
milligrams per milliliter of buffer was modified with two-armed
mPEG at 37.degree. C. Two-armed mPEG of the invention as described
was added at a molar ratio of polymer to protein amino groups of
3.3:1.
The polymer and enzyme conjugates were purified by ultrafiltration
and concentrated in an Amicon system with a PM 10 membrane (cut off
10,000) to eliminate N-hydroxysuccinimide and reduce polymer
concentration. The conjugates were further purified from the excess
of unreacted polymer by gel filtration chromatography on a
Pharmacia Superose 12 column, operated by an FPLC instrument, using
10 mM phosphate buffer of pH 7.2, 0.15 M in NaCl, as eluent.
Protein concentration for the native forms of ribonuclease,
catalase, and trypsin was evaluated spectrophotometrically using
molar extinction coefficients of 945.times.10.sup.3 M.sup.-1
cm.sup.-1, 1.67.times.10.sup.5 M.sup.-1 cm.sup.-1 and
3.7.times.10.sup.4 M.sup.-1 cm.sup.-1 at 280 nm, respectively. The
concentration of native asparaginase was evaluated by biuret assay.
Biuret assay was also used to evaluate concentrations of the
protein modified forms.
The extent of protein modification was evaluated by one of three
methods. The first is a colorimetric method described in Habeeb, A.
F. S. A. (1966) Determination of free amino groups in protein by
trinitrobenzensulphonic acid. Anal. Biochem. 14, 328-336. The
second method is amino acid analysis after acid hydrolysis. This
method was accomplished by two procedures: 1) the post-column
procedure of Benson, J. V., Gordon, M. J., and Patterson, J. A.
(1967) Accelerated chromatographic analysis of amino acid in
physioloaica fluids containing vitamin and asparaqine. Anal. Biol.
Chem. 18, 288-333, and 2) pre-column derivatization by
phenylisothiocyanate (PITC) according to Bidlingmeyer, B. A.,
Cohen, S. A., and Tarvin, T. L. (1984) Rapid analysis of amino
acids using pre-column derivatization. J. Chromatography 336,
93-104.
The amount of bound linear mPEG was evaluated from norleucine
content with respect to other protein amino acids. The amount of
two-armed, mPEG-disubstituted lysine was determined from the
increase in lysine content. One additional lysine is present in the
hydrolysate for each bound polymer.
III. Analysis of Polymer and Enzyme Conjugates
Five different model enzymen, ribonuclease, catalase, asparaginase,
trypsin and uricase, were modified with linear, conventional mPEG
of molecular weight 5000 having a norleucine amino acid spacer arm
activated as succinimidl ester and with a two-armed,
mPEG-disubstituted lysine of the invention prepared from the same
linear, conventional mPEG as described above in connection with the
two step procedure. The molecular weight of the two-armed mPEG
disubstituted lysine of the invention was approximately 10,000.
A. Comparison of Enzyme Activity. The catalytic properties of the
modified enzymes were determined and compared and the results are
presented in Table 1 below. To facilitate comparison, each enzyme
was modified with the two polymers to a similar extent by a careful
choice of polymer to enzyme ratios and reaction temperature.
Ribonuclease with 50% and 55% of the amino groups modified with
linear mPEG and two-armed mPEG, respectively, presented 86% and 94%
residual activity with respect to the native enzyme. Catalase was
modified with linear mPEG and with two-armed mPEG to obtain 43% and
38% modification of protein amino groups, respectively. Enzyme
activity was not significantly changed after modification. Trypsin
modification was at the level of 50% and 57% of amino groups with
linear mPEG and with two-armed mPEG, respectively. Esterolytic
activity for enzyme modified with linear mPEG and two-armed mPEG,
assayed on the small substrate TAME, was increased by the
modification co 120% and 125%, respectively. Asparaginase with 53%
and 40% modified protein amino groups was obtained by coupling with
linear mPEG and two-armed mPEG, respectively. Enzymatic activity
was increased, relative to the free enzyme, to 110% for the linear
mPEG conjugate and to 133% for the two-armed mPEG conjugate.
While not wishing to be bound by theory, it is possible that in the
case of trypsin and asparaginase, that modification produces a more
active form of the enzyme. The K.sub.m values of the modified and
unmodified forms are similar.
For the enzyme uricase a particularly dramatic result was obtained.
Modification of uricase with linear mPEG resulted in total loss of
activity. While not wishing to be bound by theory, it is believed
that the linear mPEG attached to an amino acid such as lysine that
is critical for activity. In direct contrast, modification of 40%
of the lysines of uricase with two-armed mPEG gave a conjugate
retaining 70% activity.
It is apparent that modification of enzymes with two-armed mPEG
gives conjugates of equal or greater activity than those produced
by conventional linear mPEG modification with monosubstituted
structures, despite the fact that two-armed mPEG modification
attaches twice as much polymer to the enzyme.
Coupling two-armed mPEG to asparaginase with chlorotriazine
activation as described in the background of the invention gave
major loss of activity. Presumably the greater activity of enzymes
modified with a two-armed mPEG of the invention results because the
bulky two-armed mPEG structure is less likely than monosubstituted
linear mPEG structures to penetrate into active sites of the
proteins.
TABLE-US-00001 TABLE 1 Properties of enzymes modified by linear
mPEG and two-armed mPEG. NH.sub.2:POLYMER % % ENZYME.sup.a MOLAR
RATIO MODIFICATION ACTIVITY Km (M) Kcas (min.sup.-1) Ribonuclease
RN 1:0 0 100 RP1 1:2.5 50 86 RP2 1:5 55 94 Catalase CN 1:0 0 100 .
CP1 1:5 43 100 CP2 1:10 38 90 Trypsin.sup.b TN 1:0 0 100 8.2
.times. 10.sup.-5 830 TP1 1:2.5 50 120 7.6 .times. 10.sup.-5 1790
TP2 1:2.5 57 125 8.0 .times. 10.sup.-5 2310 Asparaginase AN 1:0 0
100 3.31 .times. 10.sup.-6 523 AP1 1:3 53 110 3.33 .times.
10.sup.-6 710 AP2 1:3.3 40 133 3.30 .times. 10.sup.-6 780 Uricase
UP 1:0 0 100 UP1 1:5 45 0 UP2 1:10 40 70 .sup.aN = native enzyme,
P1 = enzyme modified with linear mPEG, P2 = enzyme modified with
two-armed mPEG. .sup.bFor trypsin only the esterolytic activity is
reported.
Enzymatic activity of native and modified enzyme was evaluated by
the following methods. For ribonuclease, the method was used of
Crook, E. M., Mathias, A. P., and Rabin, B. R. (1960)
Spectrophotometric assay of bovine pancreatic ribonuclease by the
use of cytidine 2':3' phosphate. Biochem. J. 74, 234-238. Catalase
activity was determined by the method of Beers, R. F. and Sizer, I.
W. (1952) A spectrophotometric method for measuring the breakdown
of hydrogen peroxide by catalase. J. Biol. Chem. 195, 133-140. The
esterolytic activity of trypsin and its derivatives was determined
by the method of Laskowski, M. (1955) Trypsinogen and trypsin.
Methods Enzymol. 2, 26-36. Native and modified asparaginase were
assayed according to a method reported by Cooney, D. A., Capizzi,
R. L. and Handschumacher, R. E. (1970) Evaluation of L-asparagine
metabolism in animals and, man. Cancer Res. 30, 929-935. In this
method, 1.1 ml containing 120 .mu.g of .alpha.-ketoglutaric acid,
20 Ul of glutamic-oxalacetic transaminase, 30 Ul of malate
dehydrogenase, 100 .mu.g of NADH, 0.5 .mu.g of asparaginase and 10
.mu.moles of asparagine were incubated in 0.122 M Tris buffer, pH
8.35, while the NADH absorbance decrease at 340 nm was
followed.
B. Proteolytic Digestion of Free Enzyme and Conjugates. The rates
at which proteolytic enzymes digest and destroy proteins was
determined and compared for free enzyme, enzyme modified by
attachment of linear activated mPEG, and enzyme modified by
attachment of an activated two-armed mPEG of the invention. The
proteolytic activities of the conjugates were assayed according to
the method of Zwilling, R., and Neurath, H. (1981) Invertrebate
protease. Methods Enzymol. 80, 633-664. Four enzymes were used:
ribonuclease, catalase, trypsin, and asparaginase. From each enzyme
solution, aliquots were taken at various time intervals and enzyme
activity was assayed spectrophotometrically.
Proteolytic digestion was performed in 0.05 M phosphate buffer of
pH 7.0. The free enzyme, linear mPEG and protein conjugate, and
two-armed mPEG-protein conjugates were exposed to the known
proteolytic enzymes trypsin, pronase, elastase or subtilisin under
conditions as follows.
For native ribonuclease and its linear and two-armed mPEG
conjugates, 0.57 mg protein was digested at room temperature with
2.85 mg of pronase, or 5.7 mg of elastase, or with 0.57 mg of
subtilisin in a total volume of 1 ml. Ribonuclease with 50% and 55%
of the amino groups modified with linear mPEG and two-armed mPEG,
respectively, was studied for stability to proteolytic digestion by
pronase (FIG. 1(a)), elastase (FIG. 1(b)) and subtilisin (FIG.
1(c)). Polymer modification greatly increases the stability to
digestion by all three proteolytic enzymes, but the protection
offered by two-armed mPEG is much more effective as compared to
linear mPEG.
For native and linear and two-armed mPEG-modified catalase, 0.58 mg
of protein were digested at room temperature with 0.58 mg of
trypsin or 3.48 mg of pronase in a total volume of 1 ml. Catalase
was modified with linear mPEG and two-armed mPEG to obtain 43% and
38% modification of protein amino groups, respectively. Proteolytic
stability was much greater for the two-armed mPEG derivative than
for the monosubstituted mPEG derivative, particularly toward
pronase (FIG. 3(a)) and trypsin (FIG. 3(b)), where no digestion
took place.
Autolysis of trypsin and its linear and two-armed mPEG derivatives
at 37.degree. C. was evaluated by esterolytic activity of protein
solutions at 25 mg/ml of TAME. Trypsin modification was at the
level of 50% and 57% of amino groups with linear mPEG and two-armed
mPEG, respectively. Modification with linear mPEG and two-armed
mPEG reduced proteolytic activity of trypsin towards casein, a high
molecular weight substrate: activity relative to the native enzyme
was found, after 20 minutes incubation, to be 64V for the linear
mPEG and protein conjugate and only 35% for the two-armed mPEG
conjugate. In agreement with these results, the trypsin autolysis
rate (i.e., the rate at which trypsin digests trypsin), evaluated
by enzyme esterolytic activity, was totally prevented in two-armed
mPEG-trypsin but only reduced in the linear mPEG-trypsin conjugate.
To prevent autolysis with linear mPEG, modification of 78% of the
available protein amino groups was required.
For native and linear mPEG- and two-armed mPEG-modified
asparaginase, 2.5 .mu.g were digested at 37.degree. C. with 0.75 mg
of trypsin in a total volume of 1 ml. Asparaginase with 53% and 40%
modified protein amino groups was obtained by coupling with linear
mPEG and two-armed mPEG, respectively. Modification with two-armed
mPEG had an impressive influence on stability towards proteolytic
enzyme. Increased protection was achieved at a lower extent of
modification with respect to the derivative obtained with the
two-armed polymer (FIG. 5).
These data clearly show that two-armed mPEG coupling is much more
effective than conventional linear mPEG coupling in providing a
protein with protection against proteolysis. While not wishing to
be bound by theory, it is believed that the two-armed mPEG, having
two polymer chains bound to the same site, presents increased
hindrance to approaching macromolecules in comparison to linear
mPEG.
C. Reduction of Protein Antigenicity. Protein can provoke an immune
response when injected into the bloodstream. Reduction of protein
immunogenicity by modification with linear and two-armed mPEG was
determined and compared for the enzyme superoxidedismutase
("SOD").
Anti-SOD antibodies were obtained from rabbit and purified by
affinity chromatography. The antigens (SOD, linear mPEG-SOD, and
two-armed mPEG-SOD) were labelled with tritiated succinimidyl
propionate to facilitate tracing. Reaction of antigen and antibody
were evaluated by radioactive counting. In a 500 .mu.L sample, the
antigen (in the range of 0-3 .mu.g) was incubated with 2.5 .mu.g of
antibody. The results show the practical disappearance of antibody
recognition for two-armed mPEG-SOD, while an appreciable
antibody-antigen complex was formed for linear mPEG-SOD and native
SOD.
D. Blood Clearance Times. Increased blood circulation half lives
are of enormous pharmaceutical importance. The degree to which mPEG
conjugation of proteins reduces kidney clearance of proteins from
the blood was determined and compared for free protein, protein
modified by attachment of conventional, linear activated mPEG, and
protein modified by attachment of the activated two-armed mPEG of
the invention. Two proteins were used. These experiments were
conducted by assaying blood of mice for the presence of the
protein.
For linear mPEG-uricase and two-armed mPEG-uricase, with 40%
modification of lysine groups, the half life for blood clearance
was 200 and 350 minutes, respectively. For unmodified uricase the
result was 50 minutes.
For asparaginase, with 53% modification with mPEG and 40%
modification with two armed mPEG, the half lives for blood
clearance were 1300 and 2600 minutes, respectively. For unmodified
asparaginase the result was 27 minutes.
E. Thermal Stability of Free and Conjugated Enzymes. Thermal
stability of native ribonuclease, catalase and asparaginase and
their linear mPEG and two-armed mPEG conjugates was evaluated in
0.5 M phosphate buffer pH 7.0 at 1 mg/ml, 9 .mu.g/ml and 0.2 mg/ml
respectively. The samples were incubated at the specified
temperatures for 15 min., 10 min., and 15 min, respectively, cooled
to room temperature and assayed spectrophotometrically for
activity.
Increased thermostability was found far the modified forms of
ribonuclease, as shown in FIG. 2, at pH 7.0, after 15 min.
incubation at different temperatures, but no significant difference
between the two polymers was observed. Data for catalase, not
reported here, showed that modification did not influence catalase
thermostability. A limited increase in thermal stability of linear
and two-armed mPEG-modified asparaginase was also noted, but is not
reported.
F. pH Stability of the Free and Conjugated Enzymes. Unmodified and
polymer-modified enzymes were incubated for 20 hrs in the following
buffers: sodium acetate 0.05 M at a pH of from 4.0 to 6.0, sodium
phosphate 0.05 M at pH 7.0 and sodium borate 0.05 M at a pH of from
8.0 to 11. The enzyme concentrations were 1 mg/ml, 9 .mu.g/ml, 5
.mu.g/ml for ribonuclease, catalase, and asparaginase respectively.
The stability to incubation at various pH was evaluated on the
basis of enzyme activity.
As shown in FIG. 2b, a decrease in pH stability at acid and alkline
pH values was found for the linear and two-armed mPEG-modified
ribonuclease forms as compared to the native enzyme. As shown in
FIG. 4, stability of the linear mPEG and two-armed mPEG conjugates
with catalase was improved for incubation at low pH as compared to
native catalase. However, the two-armed mPEG and linear mPEG
conjugates showed equivalent pH stability. A limited increase in pH
stability at acid and alkaline pH values was noted for linear and
two-armed mPEG-modified asparaginase as compared to the native
enzyme.
It should be recognized that there are thousands of proteins and
enzymes that can be usefully modified by attachment to the polymer
derivatives of the invention. Proteins and enzymes can be derived
from animal sources, humans, microorganisms, and plants and can be
produced by genetic engineering or synthesis. Representatives
include: cytokines such as various interferons (e.g.
interferon-.alpha., interferon-.beta., interferon-.gamma.),
interleukin-2 and interleukin-3), hormones such as insulin, growth
hormone-releasing factor (GRF), calcitonin, calcitonin gene related
peptide (CGRP), atrial natriuretic peptide (ANP), vasopressin,
corticortropin-releasing factor (CRF), vasoactive intestinal
peptide (VIP), secretin, .alpha.-melanocyte-stimulating hormone
(.alpha.-MSH), adrenocorticotropic hormone (ACTH), cholecystokinin
(CCK), glucagon, parathyroid hormone (PTH), somatostatin,
endothelin, substance P, dynorphin, oxytocin and growth
hormone-releasing peptide, tumor necrosis factor binding protein,
growth factors such as growth hormone (GH), insulin-like growth
factor (IGF-I, IGF-II), .beta.-nerve growth factor (.beta.-NGF),
basic fibroblast growth factor (bFGF), transforming growth factor,
erythropoietin, granulocyte colony-stimulating factor (G-CSF),
granulocyte macrophage colony-stimulating factor (GM-CSF),
platelet-derived growth factor (PDGF) and epidermal growth factor
(EGF), enzymes such as tissue plasminogen activator (t-PA),
elastase, superoxide dismutase (SOD), bilirubin oxydase, catalase,
uricase and asparaginase, other proteins such as ubiquitin, islet
activating protein (LAP), serum thymic factor (STP), peptiae-T and
trypsin inhibitor, and derivatives thereof. In addition to protein
modification, the two-armed polymer derivative of the invention has
a variety of related applications. Small molecules attached to
two-armed activated mPEG derivatives of the invention can be
expected to show enhanced solubility in either aqueous or organic
solvents. Lipids and liposomes attached to the derivative of the
invention can be expected to show long blood circulation lifetimes.
Other particles than lipids and surfaces having the derivative of
the invention attached can be expected to show nonfouling
characteristics and to be useful as biomaterials having increased
blood compatibility and avoidance of protein adsorption.
Polymer-ligand conjugates can be prepared that are useful in two
phase affinity partitioning. The polymers of the invention could be
attached to various forms of drugs to produce prodrugs. Small drugs
having the multisubstituted derivative attached can be expected to
show altered solubility, clearance time, targeting, and other
properties.
The invention claimed herein has been described with respect to
particular exemplified embodiments. However, the foregoing
description is not intended to limit the invention to the
exemplified embodiments, and the skilled artisan should recognize
that variations can be made within the scope and spirit of the
invention as described in the foregoing specification. The
invention includes all alternatives, modifications, and equivalents
that may be included within the true spirit and scope of the
invention as defined by the appended claims.
* * * * *